Cell prep and gradient centrifugation are not appropriate for small (microgram) amounts. Better using elution from 4%PAGE but do not forget to add BSA and NP40 in the gel elution buffer. My recommendation is to optimize the nucleosome reconstitution instead purification after reconstitution.

I agree with Dr. Angelov. Optimization of nucleosome reconstitution to get a high quality nucleosome preparation should be the key step. If you can afford purified DNA and core histones for reconstitution, I would recommend using higher amounts of DNA and core histones (up to 0.5mg/ml) for the reconstitution without addition of BSA. Then I would run gradient centrifugation if further purification is required. Good luck with your experiments!

Dimitar Angelov

Thank you for your suggestions. I tried using PAGE elution once using the "crush and soak" method, but somehow I only got DNA (it seemed like somehow my nucleosome dissociated). Do you have any suggestions for going into this route? I couldn't find any protocol in literature because it seems like this is an atypical route for purifying nucleosome instead of DNA.

Use the Widom 601 positioning DNA sequence for nuc reconstitution. The nucleosomal band of the PAGE should have at least 5-10 ug nuc (load ~ 10 ug nucleosomes/wells) on the PAGE. Put the PAGE slices into the elution buffer without crushing and do not let it dry. Put 100-200 ug/ml BSA and 0.1% NP40 in the elution buffer (10 mM Tris, 0.25 mM EDTA, 10 mM NaCl). Use silicognized eppendorf tubes. After 1.5h elution at 30°C change the buffer and elute the PAGE slice one more time. Do not use spin sephadex column.